Carboxyfullerenes localize within mitochondria and prevent the UVB‐induced intrinsic apoptotic pathway

Abstract

Carboxyfullerenes (CF) act as free radical scavengers in many cell settings and prevent apoptosis in vitro and in vivo. CF protect normal human keratinocytes from UVB-induced apoptosis, although the mechanisms underlying this effect remain to be clarified. Double-staining confocal laser microscopy revealed that CF penetrate the cell and colocalize with cytokeratin-18 within cytoplasm. This localization was confirmed by transmission electron microscopy that showed CF intermingled with keratin filaments. Moreover, double-staining with the mitochondrial marker anti-F1-ATPase antibody demonstrated that CF are expressed in mitochondria. Transmission electron microscopy confirmed that CF actually localize within mitochondria. Then, normal human keratinocytes were UVB-irradiated in the presence or absence of CF at different doses. CF protected keratinocytes from apoptosis induced by reactive oxygen species. CF scavenging effect is associated with a partial blockade of the UVB-induced intrinsic apoptotic pathway by down-modulating caspase-9 activation and cytochrome c release, and by inhibiting the down-regulation of the inhibitor of apoptosis proteins (IAP) survivin, livin, IAP-1 and IAP-2. Finally, CF prevented the cleavage of Bid, up-regulation of Bad and down-regulation of Mcl-1 induced by UVB. Taken together, these results indicate that CF penetrate human keratinocytes, localize within mitochondria where they act both by scavenging free radicals and by protecting cells from apoptosis.